Characterization of recombinant chloroperoxidase, and F103A and C29H/C79H/C87H mutants

Mechanistically and structurally chloroperoxidase (CPO) occupies a unique niche among heme containing enzymes. Chloroperoxidase catalyzes a broad range of reactions, such as oxidation of organic substrates, dismutation of hydrogen peroxide, and mono-oxygenation of organic molecules. To expand the synthetic utility of CPO and to appreciate the important interactions that lead to CPO’s exceptional properties, a site-directed mutagenesis study was undertaken. ^ Recombinant CPO and CPO mutants were heterologously expressed in Aspergillus niger. The overall protein structure was almost the same as that of wild type CPO, as determined by UV-vis, NMR and CD spectroscopies. Phenylalanine103, which was proposed to regulate substrate access to the active site by restricting the size of substrates and to control CPO’s enantioselectivity, was mutated to Ala. The ligand binding affinity and most importantly the catalytic activity of F103A was dramatically different from wild type CPO. The mutation essentially eliminated the chlorination and dismutation activities but enhanced, 4-10 fold, the epoxidation, peroxidation, and N-demethylation activities. As expected, the F103A mutant displayed dramatically improved epoxidation activity for larger, more branched styrene derivatives. Furthermore, F103A showed a distinctive enantioselectivity profile: losing enantioselectivity to styrene and cis-β-methylstyrene; having a different configuration preference on α-methylstyrene; showing higher enantioselectivites and conversion rates on larger, more branched substrates. Our results show that F103 acts as a switch box that controls the catalytic activity, substrate specificity, and product enantioselectivity of CPO. Given that no other mutant of CPO has displayed distinct properties, the results with F103A are dramatic. ^ The diverse catalytic activity of CPO has long been attributed to the presence of the proximal thiolate ligand. Surprisingly, a recent report on a C29H mutant suggested otherwise. A new CPO triple mutant C29H/C79H/C87H was prepared, in which all the cysteines were replaced by histidine to eliminate the possibility of cysteine coordinating to the heme. No active form protein was isolated, although, successful transformation and transcription was confirmed. The result suggests that Cys79 and Cys87 are critical to maintaining the structural scaffold of CPO. ^ In vitro biodegradation of nanotubes by CPO were examined by scanning electron microscope method, but little oxidation was observed. ^

Mechanisms of chloroperoxidase-catalyzed enantioselective reactions as probed by site-directed mutagenesis and isotopic labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in k cat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

The role of protein oxidation in ageing & disease

Ageing is a natural phenomenon of the human lifecycle, yet it is still not understood what causes the deterioration of the human body near the end of the lifespan. One popular theory is the Free Radical Theory of Ageing, which proposes that oxidative damage to biomolecules causes ageing of tissues. The ageing population is affected by many chronic diseases. This study focused on sarcopenia (muscle loss in ageing) and obesity as two models for comparison of oxidative damage in muscle proteins in mice. The aim of the study was to develop advanced mass spectrometry methods to detect specific oxidative modifications to mouse muscle proteins, including oxidation, nitration, chlorination, and carbonyl group formation, but western blotting was also used to provide complementary information on the oxidative state of proteins from aged and obese muscle. Mass spectrometry proved to be a powerful tool, enabling identification of the types of modifications present, the sites at which they were present and percentage of the peptide populations that were modified. Targeted and semi-targeted mass spectrometry methods were optimised for the identification and quantitation of the oxidised residues in muscle proteins. The development of the quantitative methods enabled comparisons of mass spectrometry instruments. Both the Time of Flight and QTRAP systems showed advantages of using the different mass analysers to quantify oxidative modifications. Several oxidised residues were characterised and quantified in both the obese and sarcopenic models, and higher levels of oxidation were found compared to their control counterparts. Residues found to be oxidised were oxidation of proline, tyrosine and tryptophan, dioxidation of methionine, allysine and nitration of tyrosine. However quantification was performed on methionine dioxidation and cysteine trioxidation containing residues in SERCA. The combination of measuring residue susceptibility and functional studies could contribute to understanding the overall role of oxidation in ageing and obesity.

Estudo da produção eletroquímica de espécies de cloro ativo para tratamento de efluentes sintéticos

The treatment of wastewater is essential to human health. One of the most important steps is the disinfection treatment which uses chlorine to eliminate bacteria as required by environmental agencies. However, the identification of potentially toxic byproducts generated by this method, such as trihalomethanes, has stimulated the development of new alternative disinfection technologies. Among them, heterogeneous photocatalysis, TiO2 photocatalysis and electrochemical disinfection are considered suitable alternatives to the chlorination method. Thus, the present dissertation analyzes the evolution of active chlorine species in a synthetic NaCl solution and it is tested to treat a synthetic solution of the dye Reactive Blue 19 using boron-doped diamond (BDD) and ruthenium oxide (Ti/Ru0.3Ti0.7O2) as anodes. The indirect electrochemical process was discussed in terms of mineralization of the total organic load and percentage of color removal in order to evaluate the applicability of electrochemical technology. Electrochemical experiments were carried out with different current densities (25, 50 and 75 mA.cm-2) during 120 minutes. On the other hand, other important parameter in this study was the influence of the proportion sp3/sp2 on BDD anode on the performance of the evolution of active chlorine species which was investigated by electrolytic techniques (linear polarization), with the intention of determining the related training oxidizing species and consumption energy to chemical or electrochemical reactions. From the results, it can be noted that the BDD electrode showed better efficiency throughout the electrochemical process.

Estudo da produção eletroquímica de espécies de cloro ativo para tratamento de efluentes sintéticos

The treatment of wastewater is essential to human health. One of the most important steps is the disinfection treatment which uses chlorine to eliminate bacteria as required by environmental agencies. However, the identification of potentially toxic byproducts generated by this method, such as trihalomethanes, has stimulated the development of new alternative disinfection technologies. Among them, heterogeneous photocatalysis, TiO2 photocatalysis and electrochemical disinfection are considered suitable alternatives to the chlorination method. Thus, the present dissertation analyzes the evolution of active chlorine species in a synthetic NaCl solution and it is tested to treat a synthetic solution of the dye Reactive Blue 19 using boron-doped diamond (BDD) and ruthenium oxide (Ti/Ru0.3Ti0.7O2) as anodes. The indirect electrochemical process was discussed in terms of mineralization of the total organic load and percentage of color removal in order to evaluate the applicability of electrochemical technology. Electrochemical experiments were carried out with different current densities (25, 50 and 75 mA.cm-2) during 120 minutes. On the other hand, other important parameter in this study was the influence of the proportion sp3/sp2 on BDD anode on the performance of the evolution of active chlorine species which was investigated by electrolytic techniques (linear polarization), with the intention of determining the related training oxidizing species and consumption energy to chemical or electrochemical reactions. From the results, it can be noted that the BDD electrode showed better efficiency throughout the electrochemical process.

(Table 3, page 8) Chemical composition and physical cahracteristics of manganese nodules from the Pacific Ocean

This paper reviews the state of the art in processing and extraction of ocean floor manganese nodules. It briefly reviews the mining sites where the abundant rich nodules occur and also discusses the metal distribution in nodules in view of economical processing and extraction of these metal values. The paper discloses in a detailed manner the physical and chemical characteristics of nodules, including porosity, surface area, water content and the effect of temperature on crystal structure of major constituents of nodules. In the extraction aspect of nodules, the paper reviews two different extraction schemes revealed in the literature, namely hydrometallurgical treatment and pyrometallurgical treatment. The hydrometallurgical treatments include acid leaching, ammonia leaching, leaching with reducing agents and leaching after high temperature pre-treatments such as in sulfating rousting, while the pyrometallurgical processes include smelting, chlorination-vaporization and segregation. The paper also covers metal recovery processes from leach liquor. An economic survey of processing nodules has been made in terms of problems associated with metal-marketing, and impact of metal production from nodules on mineral industries.

Preparation of low protein latex : Effect of different cure systems and fillers on its properties

Natural rubber latex, an aqueous colloidal dispersion of polyisoprene is widely used in production of gloves, catherers, rubber bands etc. The natural rubber latex content present in products such as gloves causes allergic problems. Of the different types of allergies reported, latex is known to produce Type I and Type IV allergies. Type I is called immediate hypersensitivity and type IV is called delayed hypersensitivity. It has been reported that some of the proteins present in the latex are mainly responsible for the allergic reactions type I. Significant reduction in the allergic response (type I) of natural rubber latex can be achieved by the reduction in its protein content, however out of the total proteins present in the latex or latex film only a fraction is extractable. The major techniques employed to reduce protein content of latex include leaching, autoclaving, chlorination, use of proteolytic enzymes and use of non ionic surfactants. Sulphur vulcanization of dipped products is responsible for Type IV allergy. N-nitrosamine, a carcinogenic substance is produced as a result of sulphur vulcanization. Radiation vulcanization can be used as an alternative for sulphur vulcanization. The current research deals with techniques to reduce the allergy associated with latex products. To reduce the type I allergy, low protein latex is developed using polyethylene glycol, a non- ionic surfactant. The present study employs radiation vulcanization to eliminate type IV allergy. The effect of different cure systems and fillers on the properties of low protein latex is also investigated as a part of the study.

Influence of the metal precursor on the catalytic behavior of Pt/Ceria catalysts in the preferential oxidation of CO in the presence of H2 (PROX)

The effect of the metal precursor (presence or absence of chlorine) on the preferential oxidation of CO in the presence of H2 over Pt/CeO2 catalysts has been studied. The catalysts are prepared using (Pt(NH3)4)(NO3)2 and H2PtCl6, as precursors, in order to ascertain the effect of the chlorine species on the chemical properties of the support and on the catalytic behavior of these systems in the PROX reaction. The results show that chloride species exert an important effect on the redox properties of the oxide support due to surface chlorination. Consequently, the chlorinated catalyst exhibits a poorer catalytic activity at low temperatures compared with the chlorine-free catalyst, and this is accompanied by a higher selectivity to CO2 even at high reaction temperatures. It is proposed that the CO oxidation mechanism follows different pathways on each catalyst.

Mechanisms of Chloroperoxidase-catalyzed Enantioselective Reactions as Probed by Site-directed Mutagenesis and Isotopic Labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in kcat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Effect of gamma-irradiation on total organic carbon and trihalomethane formation potential

This research was conducted to study the use of radiation in water treatment as an alternative to chlorination which has caused health concerns due to the formation of harmful disinfection by-products. Groundwater solutions from the Biscayne aquifer were radiated with Cobalt-60 gamma radiation and studied for changes in dissolved organic carbon (DOC), UV absorbance at 254 nm (UV254), fluorescence and trihalomethane formation potential (THMFP). Molecular fractionations were conducted by ultrafiltration. Effect of the combination of radiation/peroxide was studied for DOC and UV254. Radiation showed significant removal in DOC and THMFP. Similar results were seen in the fluorescence and UV absorbance experiments. Radiation/peroxide did not improve the DOC removal. Radiation of the groundwater samples broke the larger molecular weight fractions in to smaller fractions.